EP4116408A1 - Procédé de culture sans méthanol de levure méthylotrophes pour la biosynthèse de produits à valeur ajoutée - Google Patents

Procédé de culture sans méthanol de levure méthylotrophes pour la biosynthèse de produits à valeur ajoutée Download PDF

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EP4116408A1
EP4116408A1 EP22180001.4A EP22180001A EP4116408A1 EP 4116408 A1 EP4116408 A1 EP 4116408A1 EP 22180001 A EP22180001 A EP 22180001A EP 4116408 A1 EP4116408 A1 EP 4116408A1
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promoter
methylotrophic yeast
carbon source
yeast cell
cell culture
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Oleg TYURIN
Mingyang Sun
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Bioboost Synbio Consulting Inc
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Bioboost Synbio Consulting Inc
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Priority claimed from US17/478,241 external-priority patent/US11512336B1/en
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/78—Hansenula
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the field of culturing/fermentation of methylotrophic yeast (e.g. Pichia pastoris or Komagataella phaffii ) to produce value added products like recombinant proteins or small molecule compounds.
  • methylotrophic yeast e.g. Pichia pastoris or Komagataella phaffii
  • Methylotrophic yeast such as Pichia pastoris, also known as Komagataella phaffii , is widely used as a host organism for recombinant protein production.
  • the ability of this yeast to use the cheap substrate methanol as sole carbon source, its high cell density fermentation capability, its secretory machinery and powerful and tightly regulated promoters have resulted in its extensive application in biotechnology.
  • GAP Glyceraldehyde-3-P dehydrogenase
  • TEF1 promoter Translation elongation factor
  • inducible promoters like AOX1 promoter (Alcohol oxidase) have advantages for production purposes as they allow biomass growth without product formation.
  • AOX1 gene in P. pastoris cells is dramatically upregulated in response to methanol, whereas it stays tightly repressed when glucose or glycerol or any other fermentable carbon source is in the media. Therefore, amongst all inducible promoters, AOX1 promoter is most abundantly used.
  • a typical bioprocess driven by any inducible promoter comprises biomass growth on glucose or glycerol (batch) phase followed by induction of expression followed by recombinant protein(s)/compound(s) production upon switching to methanol (fed-batch and induction phase).
  • inducible promoters In response to different carbon sources, all inducible promoters, including AOX1, have three regulated states of gene expression: catabolite repression (or just repression), derepression, and activation (induction). For AOX1 promoters, these states are well described, unlike for the other inducible promoters described herein.
  • glycerol or glucose or ethanol or any other fermentable carbon source When those carbon sources are depleted, the AOX1 promoter is derepressed, which means it is activated at roughly 2-5% of its methanol induction level.
  • AOX1 promoter can also be de-repressed when culture grows on so-called non-repressible carbon sources (e.g.
  • AOX1 promoter When the culture starts to consume methanol as a carbon source, with or without non-repressible carbon sources, AOX1 promoter, and the other listed promoters are fully activated (i.e. induced), Although methanol is widely used as an inducer for the AOX1 driven induction system, many shortcomings of using methanol in the fermentation process (e.g. it's flammability and toxicity) greatly limits the feasibility of this system at large industrial scales. Recently it was shown that the salts of formic acid (formates) can induce AOX1 promoter almost as well as methanol. However, it still remains unknown whether other native promoters discussed herein can be induced by formic acid or formates. Also, it has not been determined if formates or formic acid can be used as an inducer in combination with non-repressible carbon sources.
  • the safety aspect of the fermentation / culturing process is supposed to be the subject of particular attention.
  • the storage of large volumes of hazardous and flammable methanol at industrial facilities is highly undesirable.
  • the expenses for extra safety measures can add an extra 15% to total production cost.
  • methanol metabolism leads to an increase in heat evolution, which is not technologically favorable because the culturing consumes a lot of energy to chill the bioreactors.
  • Another downside of methanol metabolism is a high oxygen consumption by the culture, which is considered as a hazard because it requires a production facility with a high oxygen capacity.
  • Described herein is a culturing method that combines the use of the previously described alternative inducing agent, salts of formic acid (formates) or formic acid together with any suitable non-repressing feeding substrate, such as sorbitol, mannitol, trehalose or alanine.
  • salts of formic acid (formates) or formic acid as an alternative to methanol inducer with or without non-repressing carbon sources for the following promoters: the NAD + -dependent formate dehydrogenase (FDH) promoter (one example of which is provided as SEQ ID NO: 1), the alcohol oxidase 2 (AOX2) promoter (one example of which is provided as SEQ ID NO:2), peroxin Pex14p (PEX14) promoter (one example of which is provided as SEQ ID NO:3), the dihydroxyacetone kinase (DAK) promoter (one example of which is provided as SEQ NO:ID 5), the dihydroxyacetone synthase 1,2 (DAS1,2) promoter (example of which are provided as SEQ ID NO:10 SEQ ID NO: 11 respectively), the formyl-glutathione dehydrogenase (FGH) promoter (one example of which is provided as SEQ ID NO:4), the formaldehyde
  • a method for producing a transgenic cell product comprising:
  • a method for producing a transgenic cell product comprising:
  • an element means one element or more than one element.
  • compositions and methods include the recited elements, but do not exclude others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • Consisting of' shall mean excluding more than a trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention.
  • polynucleotide refers to a double-stranded or single-stranded DNA, as well as complementary nucleic acid sequences.
  • Polynucleotide includes a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof.
  • the nucleic acid sequences of the present disclosure may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases.
  • proteins and polypeptides refers to a sequence of amino acid residues encoded by a nucleic acid molecule.
  • a polypeptide of the disclosure may in one embodiment include various structural forms of the primary protein.
  • a polypeptide of the disclosure may be in the form of acidic or basic salts or in neutral form.
  • individual amino acid residues may be modified by oxidation or reduction.
  • the proteins and polypeptides of the present disclosure may also include truncations, analogs, and homologs of the proteins and polypeptides as described herein having substantially the same function as the proteins or polypeptides of the present disclosure.
  • construct refers to an artificially created nucleic acid, comprising a delivery vector and a gene(s) of interest, for example a vector comprising a polynucleotide described herein.
  • the polynucleotide of interest can be cloned into a plasmid of interest to produce a construct.
  • the vector is an expression vector. Possible expression vectors include but are not limited to cosmids or plasmids, so long as the vector is compatible with the host cell used.
  • the expression vectors are suitable for transformation of a host cell, which means that the expression vectors contain a polynucleotide such as those exemplified in the application and regulatory sequences selected on the basis of the host cells to confer the expression of a gene of interest.
  • Operatively linked is intended to mean that the gene of interest is linked to regulatory sequences in a manner which allows expression of this gene of interest.
  • the isolated and/or purified nucleic acid molecules, polynucleotides or vectors, constructs, or in vitro expression systems comprising these isolated and/or purified nucleic acid molecules may be used to create transgenic or recombinant organisms or recombinant cells (e.g. optionally cells of recombinant organisms) that produce polypeptides or any small molecule compound.
  • a nucleotide sequence is associated in a manner of receiving, for example, a promoter is operably linked to a coding sequence of a recombinant gene when it can affect the expression of the coding sequence.
  • the expression system comprises a recombinant vector or a part thereof as disclosed herein.
  • the expression system comprises a suitable host cell, for example, a microbial cell, a yeast cell, a plant cell, or an animal cell.
  • the host expression system comprises a yeast cell.
  • the yeast cell comprises one or more of Pichia pastoris, Komagataella kurtzmanii, Komagataella phaffii, Pichia angusta, Pichia guillermordii, Pichia methanolica, Pichia inositovera, Hansenula polymorpha, Candida boidinii, and Yarrowia lipolytica.
  • batch phase refers to the first phase of culturing / fermentation upon inoculation where the culture grows to reach the needed optical density (OD 600 ) before induction phase.
  • OD 600 optical density
  • the specific OD 600 will depend on several factors, including but by no means limited to the transgenic cell product of interest, for example, a foreign peptide or small molecule, being produced; and the host cell being used.
  • induction phase or “fed-batch phase” or “continuous phase” refer to the second phase of culturing following the batch phase, where the culture is induced, which can be considered as switching on the expression of gene(s) of interest by adding a compound called an inducer.
  • a method for producing a transgenic cell product comprising:
  • the inducible promoter may be selected from the group consisting of: NAD+-dependent formate dehydrogenase (FDH) promoter (one example of which is provided as SEQ ID NO:1); alcohol oxidase 2 (AOX2) promoter (one example of which is provided as SEQ ID NO:2); peroxin Pex14p (PEX14) promoter (one example of which is provided as SEQ ID NO:3); dihydroxyacetone kinase (DAK) promoter (one example of which is provided as SEQ ID NO: 5); dihydroxyacetone synthase 1,2 (DAS1,2) promoter (examples of which are provided as SEQ ID NO:10 and SEQ ID NO: 11 respectively); formyl-glutathione dehydrogenase (FGH) promoter (one example of which is provided as SEQ ID NO:4); formaldehyde dehydrogenase 1 (FLD1) promoter (one example of which is provided as SEQ ID NO:12); Fructose 1,6
  • the host cell may be a yeast cell, for example, selected from the group consisting of: Pichia pastoris, Komagataella kurtzmanii, Komagataella phaffii, Pichia angusta, Pichia guillermordii, Pichia methanolica, Pichia inositovera, Hansenula polymorpha, Candida boidinii, and Yarrowia lipolytica.
  • the yeast is Pichia pastoris.
  • the nucleic acid molecule further comprises a secretion peptide in frame with the transgenic cell product of interest, preferably upstream in the direction of transcription and translation relative to the product of interest or gene of interest.
  • the nucleic acid molecule further comprises an expression tag in frame with the transgenic cell product of interest, preferably at the C-terminus or N-terminus of the product of interest or gene of interest.
  • the suitable host cell culture density may be 250-350 g/L of culture (wet cell weight).
  • steps (c), (d) and (e) are repeated more than once.
  • the transgenic cell product of interest may be recovered from the growth media and additional non-repressing carbon source and inducer compound may be added to sustain growth of the host cell culture in batch phase so that product continue to be produced by the cells and recovered from the media.
  • the host cell culture density is determined prior to adding the induction compound so that the inducer compound is added at a concentration that is sufficient to induce the inducible promoter at that host cell culture density.
  • the non-repressing carbon source is initially added to the host cell culture in stages, for example, starting prior to exhaustion of the growth repressing carbon source so that initially the host cell culture is growing on both the repressing carbon source and the non-repressing carbon source.
  • the repressing carbon source is the major carbon source initially and the levels thereof are allowed to decrease until the non-repressing carbon source is the sole carbon source. As discussed herein, this prevents lags in growth of the host cell culture, as there is a gradual transition from the repressing carbon source to the non-repressing carbon source rather than an abrupt shift.
  • the non-repressing carbon source may be selected from the group consisting of sorbitol, mannitol, trehalose and alanine.
  • the fermentable repressing carbon source may be glycerol or glucose.
  • the expression vector or recombinant vector comprises an origin of replication that enables the vector to propagate in, for example, E.coli for amplification and cloning purposes.
  • the recombinant vector comprises selectable "marker genes", which enable the selection of host cells (both E.coli and yeast cells) transformed with a recombinant cassette of the application.
  • selectable marker genes include but are by no means limited to genes encoding for proteins such as aminoglycoside 3'-phosphotransferase which confers resistance to G418 antibiotic, or hygromycin B phosphotransferase which confers resistance to hygromycin.
  • Other suitable selectable marker genes will be readily apparent to one of skill in the art.
  • the expression vector further comprises a secretion peptide (e.g. ⁇ MF) that is for example linked or fused or in frame with the transgenic cell product of interest so that when expression of the transgenic cell product of interest is driven by the inducible promoter, the peptide or polypeptide that is produced from the resulting transcript includes a secretion peptide which directs the nascent polypeptide chain to the secretion pathway, as discussed herein.
  • a secretion peptide e.g. ⁇ MF
  • the product of the gene of interest GOI
  • the polypeptide produced by the expression vector further comprises, at the C' or N' terminal thereof, a detection tag that facilitates the detection of the protein of interest for example by means of Western Blotting.
  • a detection tag that facilitates the detection of the protein of interest for example by means of Western Blotting.
  • human influenza hemagglutinin (HA) tag, Myc tag, FLAG tag or HIS tag are examples of short peptides that can be used as a detection tag.
  • the repressing carbon source may be any fermentable carbon source, for example, but by no means limited to glycerol or glucose.
  • batch phase indicates intensive culture growth, for example, so that the host cell culture reaches high densities, e.g. 250-350 g/L of culture (wet cell weight).
  • the specific density of the host cell culture when the carbon source is switched from a repressive carbon source to a non-repressive carbon source may vary, depending on the product being expressed and the desired outcome. As such, while a lower cell density will in theory produce less protein, this may be desirable if the product or protein being produced is for example toxic to the cell or otherwise problematic to synthesize and/or recover at higher densities. Similarly, while higher cell densities may not be healthy for the culture overall, in some embodiments, this higher density may be desirable for efficient production of the product.
  • the inducer compound is degraded by formate dehydrogenase.
  • the inducer compound may be added and the transgenic cell product of interest recovered from the host cell culture multiple times, depending of course on the nature of the transgenic cell product being produced.
  • non-repressing carbon source may also be fed, either continuously or in batches, to the host cell culture, as discussed herein.
  • the disclosure provides a method for producing added value products like polypeptides or small molecule compounds by the culturing of methylotrophic yeast without use of methanol as an inducer, that is, with the proviso that no methanol is added as an inducer. Instead, the method uses a non-repressing carbon source for feeding and an alternative inducer for expression of (a) gene(s) of interest.
  • a process for producing added value compounds using methylotrophic yeast host expression system that comprises (a) nucleotide sequence(s) encoding the gene(s) of interest, comprising (i) culturing the yeast host cells in a batch phase providing a feeding for robust growth; and (ii) culturing the host expression system in a fed-batch phase providing a feeding with an alternative inducer, or (ii) culturing the host expression system in a continuous phase providing feeding in continuous fermentation regime with an alternative inducer.
  • the batch and fed-batch phases carbon source can be any carbon source except methanol.
  • the first and/or the second sources comprise one or more of glycerol, alanine, lactate, glycerol, glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, fructose, melibiose, maltose, rhamnose, mannose, mannitol, and raffinose.
  • the batch phase carbon source is glycerol.
  • the fed-batch and induction phase carbon source is sorbitol.
  • glycose, glycerol, ethanol, citrate, xylose, arabinose, fructose, melibiose, maltose, rhamnose, mannose, and raffinose belong to repressing carbon sources; whereas sorbitol, mannitol, alanine and trehalose are non-repressing carbon sources.
  • the continuous and induction phase carbon source can be any non-repressing carbon source except methanol.
  • the first and/or the second sources comprise one or more of alanine, sorbitol, mannitol.
  • the induction phase carbon source is sorbitol.
  • a promoter is a regulatory nucleotide sequence that drives expression of a gene of interest.
  • an inducer is a compound that regulates gene expression.
  • an inducer comprises one or more of formaldehyde, S-formylglutathione, S-hydroxymethyl glutathione, formic acid or any alkali metal or ammonium salt of formic acid or an alkaline earth metal salt of formic acid is used.
  • exemplary of such inducers are sodium formate, potassium formate, and ammonium formate.
  • the regulatory sequence is a promoter.
  • the promoter is a regulatory nucleotide sequence in the host cell or host expression system that drives the expression of a gene of interest.
  • the promoter is a constitutive promoter or an inducible promoter.
  • the promoter is selected from a group consisting of, the FDH promoter (NAD + -dependent formate dehydrogenase) promoter, the Alcohol oxidase 2 (AOX2) promoter, a dihydroxyacetone kinase (DAK) promoter, a Dihydroxyacetone synthase 1,2 (DAS1,2) promoter, the Formyl-glutathione dehydrogenase (FGH) promoter, the Formaldehyde dehydrogenase 1 (FLD1) promoter, the Fructose 1,6-bisphosphate aldolase (FBA) promoter, the Peroxisomal membrane signal receptor PTS1 (PEX5) promoter, the Alcohol dehydrogenase 2 (ADH2) promoter, and a Catalase (CAT) promoter.
  • FDH promoter NAD + -dependent formate dehydrogenase
  • AOX2 Alcohol oxidase 2
  • DAK dihydroxyacetone kinase
  • DAS1,2
  • the media comprising the host expression system is oxygenated.
  • the batch phase feed is provided at a rate that maintains a specific growth rate ( ⁇ ) of the host expression system in the culture to be in a range from about 0.03 h -1 to about 0.5 h -1 .
  • the fed-batch phase or continuous feed is provided at a rate that maintains a specific growth rate ( ⁇ ) of the host expression system in the culture to be in a range from about 0.0001 h -1 to about 0.465 h -1 .
  • a very fast growth rate for the host cell for example, growth during the batch phase, is considered to be about 0.3 - 0.4 H -1
  • a slow growth rate may be for example about 0.01 - 0.04 H -1 , which may be the growth rate of the cells during the induction phase.
  • the non-repressing carbon source is supplied to or present in the growth medium at a concentration or percentage that will support growth of the host cell culture at about 0.3 - 0.4 H -1 while the non-repressing carbon source is supplied to or present in the growth medium at a concentration or percentage that will support growth of the host cell culture at about 0.01 - 0.04 H -1 .
  • the batch phase and fed-batch /continuous phase are each carried out at a temperature of about 21°C to about 30°C. In one embodiment, the batch phase and fed-batch phase are each carried out at a temperature of about 25°C.
  • the polypeptide is a heterologous polypeptide.
  • the polypeptide comprises about ten or more amino acids.
  • heterologous refers to a polynucleotide, gene, polypeptide, or an enzyme not normally found in the host organism (e.g., recombinant cell). "Heterologous” also includes a native coding region, or portion thereof, that is reintroduced into the host organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the host's genome.
  • the heterologous polynucleotide or gene may be introduced into the host organism by, e.g., gene transfer, for example, by transformation or transfection.
  • a heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host.
  • Foreign genes can be conceptualized as native genes inserted into a non-native organism, or chimeric genes.
  • heterologous polypeptides are those polypeptides foreign to the host cell being utilized, such as a plant or human protein being produced by yeast or bacteria. While the heterologous polypeptide may be prokaryotic or eukaryotic, in some embodiments it is eukaryotic. In some embodiments, it is a plant or human protein or peptide. In some embodiments, it is a polypeptide (e.g., enzyme).
  • Variants and/or fragments of the polypeptides described herein may also be prepared by the methods disclosed herein.
  • activity of a polypeptide of the present invention can be determined by methods known in the art.
  • the polypeptide is human epidermal growth factor (hEGF) comprising the amino acid sequence set forth in SEQ ID NO:17 (NCBI Accession No. XP_016863338.1)
  • hEGF is a ⁇ 6.2 kDa polypeptide composed of 53 amino acid residues with three intramolecular disulfide bonds.
  • SEQ ID NO:17 NCBI Accession No. XP_016863338.1
  • hEGF human epidermal growth factor
  • hEGF is a ⁇ 6.2 kDa polypeptide composed of 53 amino acid residues with three intramolecular disulfide bonds.
  • One of its maj or biological functions is to promote the generation of new epithelial and endothelial cells, and to stimulate tissue repairs.
  • hEGF had been produced in various host systems including Escherichia coli, Saccharomyces cerevisiae and baculovirus. In E. coli, the produced hEGF tends to form inclusion bodies, which dramatically
  • the polypeptide is an extracellular superoxide dismutase [Cu-Zn] (hSOD3) comprising the amino acid sequence set forth in SEQ ID NO:18 (NCBI Accession No. NP_003093.2)
  • SOD is a ⁇ 30kDa polypeptide and reported to be a multimeric glycoprotein composed of at least four identical subunits in human extracellular fluids with heterogeneous affinity for heparin.
  • the potential demand for SOD in human healthcare is growing up; therefore, production of biological active SOD is of a great interest. Production of therapeutic proteins by genetically engineered yeasts was shown to be a cost-effective alternative to tissue cultures or purification from animal tissues.
  • the polypeptide is a human Lactoferrin (hLF) comprising the amino acid sequence set forth in SEQ ID NO:19 (NCBI Accession No. AAB60324.1) Lactoferrin (LF) is a member of the transferrin family of iron-binding glycoproteins. It was originally found in mammalian exocrine secretions and in specific granules of polymorphonuclear leukocytes.
  • the polypeptide is the receptor binding domain (RBD) of S (spike) glycoprotein of SARS-CoV-2 virus comprising the amino acid sequence set forth in SEQ ID NO:20 (PDB: 7CM4_A).
  • RBD receptor binding domain
  • S spike glycoprotein of SARS-CoV-2 virus
  • PDB amino acid sequence set forth in SEQ ID NO:20
  • the S protein is considered to be a primary target for vaccine design as well as antiviral therapeutics.
  • a polypeptide prepared by the method of the present invention can be isolated after expression by techniques known in the art, including, but not limited to, affinity chromatography, ion-exchange chromatography, antibody affinity, size-exclusion, or any other method that eliminates a substantial portion of the culture and/or cellular debris from the polypeptide.
  • the process provides a substantially purified polypeptide.
  • the isolated polypeptide can have activity similar to the corresponding native protein that it is derived from.
  • the polypeptide can be isolated in a correctly folded state or conformation, approximating that of the native protein, or can be further renatured or modified to put it into a correctly folded conformation using a variety of methods and/or reagents known in the art.
  • the host cells are Pichia pastoris ( e.g. , Komagataella spp ), Pichia angusta, Pichia guillermordii, Pichia methanolica, or Pichia inositovera.
  • the recombinant or host cell is Pichia pastoris.
  • the recombinant or host cell is a Mut S (methanol utilization slow) strain of P. pastoris KM71 and KM71h. It is of note however that Mut + strains such as GS115 may be used within the invention and use of a Mut S strain is not a requirement of the invention.
  • the host cell or recombinant cell is Hansenula polymorpha , Candida boidinii , or Yarrowia lipolytica.
  • a heterologous polynucleotide encoding the polypeptide is provided on a vector (e.g., plasmid) suitable for integration into the genome of the host cell in single or multiple copies per host cell.
  • the vector is a nucleotide sequence integrated into the genome.
  • the vector is a eukaryotic expression vector, preferably a yeast expression vector.
  • the expression vector is a cloned recombinant nucleotide sequence, such as the DNA sequence required for transcription of one or more recombinant gene(s) or peptides of interest and their mRNA translation in appropriate host organisms.
  • such expression vectors typically include one or more of an origin for autologous replication in a host cell, an appropriate marker (e.g., gene that confers resistance to antibiotics such as zeocin, kanamycin G418 or hygromycin), a restriction enzyme cleavage site, an appropriate promoter sequence and a transcription terminator, and these components are operably linked to interact with each other.
  • an appropriate marker e.g., gene that confers resistance to antibiotics such as zeocin, kanamycin G418 or hygromycin
  • expression vectors include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designated plasmids.
  • the expression vector of the present invention may be any expression vector suitable for expression of a recombinant gene in a host cell, which is selected according to the host organism.
  • regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
  • the nucleotide sequence of the gene of interest is under the control of a constitutive promoter, a promoter responsive to a carbon source which is fed during a batch phase of culturing, and/or a promoter responsive to a carbon source and to an inducer, which is fed /added during a fed-batch phase of culturing
  • the promoter is an endogenous promoter, wherein the polynucleotide encoding the polypeptide is integrated into the genome of the yeast host cell such that the endogenous promoter is operably linked to the heterologous polynucleotide, thereby capable of driving its expression.
  • the expression vector pL (SEQ ID NO:3) further comprises a secretory leader sequence effective for inducing the secretion of polypeptide from the host cell.
  • the expression vector pL (SEQ ID NO:13) further comprises the HA-tag (Hemagglutinin tag) for the routine detection by Western Blot using anti-HA-tag antibodies.
  • HA-tag Hemagglutinin tag
  • the secretory leader sequence may originate from a yeast ⁇ -factor source, for example from ⁇ MF , yeast phosphatase (PHO), heat shock proteins (HSP), as well as HSP tag repeats, invertase (SUC2) tag, OST1 tag, DDDK tag or combination thereof, or any other secretion peptide described in the literature.
  • a yeast ⁇ -factor source for example from ⁇ MF , yeast phosphatase (PHO), heat shock proteins (HSP), as well as HSP tag repeats, invertase (SUC2) tag, OST1 tag, DDDK tag or combination thereof, or any other secretion peptide described in the literature.
  • the step of growing the recombinant cell comprising the heterologous polynucleotide includes growing the cell in a medium comprising a first carbon source, for example, a fermentable or repressing carbon source.
  • a first carbon source for example, a fermentable or repressing carbon source.
  • the medium is an aqueous medium comprising the first carbon source, and optionally one or more further ingredients such as, for example, salts (e.g., phosphate and/or sulphate, and the like), antibiotics, vitamins, trace metal ions, agents to keep the pH at a desired level, phosphate salts, and/or antifoaming agents.
  • salts e.g., phosphate and/or sulphate, and the like
  • antibiotics e.g., phosphate and/or sulphate, and the like
  • vitamins e.g., phosphate and/or sulphate, and the like
  • antibiotics e.g., phosphate and/or sulphate, and the like
  • vitamins e.g., phosphate and/or sulphate, and the like
  • trace metal ions e.g., phosphate and/or sulphate, and the like
  • agents to keep the pH at a desired level e.g., phosphat
  • the medium comprises one or more of phosphoric acid, calcium sulfate, potassium sulfate, magnesium sulfate, potassium hydroxide, and glycerol.
  • the medium further comprises one or more of cupric sulfate, sodium iodide, manganese sulfate, sodium molybdate, boric acid, cobalt chloride, zinc chloride, ferrous sulfate, biotin, and sulfuric acid.
  • the batch and fed-batch carbon source (or first carbon source) comprises one or more of alanine, lactate, glycerol, glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, fructose, melibiose, maltose, rhamnose, mannose, mannitol, and raffinose.
  • the continuous and induction phases carbon source comprises one or more of alanine, sorbitol, mannitol, and trehalose.
  • methylotrophic yeast like Pichia pastoris prefers glucose or its precursors (disaccharides) so that to assimilate it through glycolysis
  • methylotrophic yeast like Pichia pastoris prefer glycerol as a carbon source assimilating it through G3P (glycerol-3-phosphate) - DHAP (dihydroxyacetone phosphate) pathway.
  • G3P glycerol-3-phosphate
  • DHAP dihydroxyacetone phosphate
  • the batch phase and/or the fed-batch carbon source are non-fermentable carbon sources.
  • the recombinant cell in a batch phase, is cultured in a saline medium with a glycerol.
  • the recombinant cell comprising the heterologous polynucleotide is grown in the medium in a fermenter, which, as used herein, also refers to for example a bioreactor or any other suitable apparatus for culturing the recombinant cells) employing a batch protocol whereby the cells are grown using the first carbon source (e.g., glycerol).
  • Cell growth may be monitored periodically and may continue until the first carbon source (e.g., glycerol) is consumed.
  • complete consumption of the first carbon source e.g., glycerol
  • DO dissolved oxygen
  • the length of time needed to consume all the first carbon source can vary depending on the density of the initial inoculum. That is, addition glycerol or other fermentable carbon source may be added in order to bring the host cell culture density to the desired density.
  • sampling of the culture to measure cell density may be performed at the end of the first carbon source (e.g., glycerol) feed stage, e.g., cell density can be measured by withdrawing a sample from e.g., the fermenter at each timepoint and using an aliquot for measuring cell density e.g., at a wavelength of 600 nm.
  • cell growth can be evaluated by measuring the wet cell weight, pH, microscopic purity, protein concentrations and/or activity.
  • the step of growing comprises adding a culture comprising the cell to the medium comprising the first carbon source.
  • an initial amount of the first carbon source in the medium is at about 4% by volume of the first carbon source.
  • a carbon source-limited (e.g., glycerol-limited) feeding phase (e.g., employing a fed-batch protocol) follows e.g., until the desired level of biomass is reached.
  • a glycerol-limited feeding phase commences until a desired level of the biomass is reached.
  • a second phase e.g., glycerol fed-batch phase
  • adding the appropriate carbon source e.g., glycerol
  • the step of growing further comprises continuously adding the first carbon source to the medium at a first feed rate from a solution comprising the first carbon source.
  • the feed rate of the fed-batch carbon source is provided at such a rate so to maintain the specific growth rate ( ⁇ ) of the culture in the range 0.001 - 0.5 h -1 .
  • the first feed rate is initiated after the initial amount of the batch phase carbon source is completely consumed by the culture.
  • the step of culturing comprises adding the fed-batch carbon source to the medium at a second feed rate and decreasing the first feed rate. As discussed herein, this provides a gradual transition from growth of the host cell culture on the repressing carbon source to growth on the non-repressing carbon source.
  • the feed rate of the continuous and/or induction phase carbon source is provided at such a rate so to maintain the specific growth rate ( ⁇ ) of the culture in the range 0,001 - 0,5 h -1 .
  • an aqueous solution comprising the second carbon source and trace salts is introduced into the medium.
  • the second carbon source (e.g., sorbitol) feed is stopped if DO cannot be maintained above 20%, then resumed when the DO increases to at least about 20%.
  • increasing agitation, aeration, pressure and/or oxygen feeding can help increase and/or maintain the DO above 20%.
  • culturing of methylotrophic yeast is carried out under aerobic conditions, so the cells are respiratory active on either of the substrates.
  • Gradual increase of DO means that the culture has not adapted to the new carbon source yet and not actively consuming it. So, adding more of the second carbon source when DO is not stabilized yet and still is in uptrend can lead to accumulation of the substrate to a stressful threshold concentration.
  • the repressing carbon source used in batch and fed-batch phases needs to be completely depleted for efficient inducing of the listed promoters to be obtained upon adding an inducing agent.
  • the inducer comprises one or more of formaldehyde, S-formylglutathione, S-hydroxymethyl glutathione, formic acid or any alkali metal salt of formic acid or an alkaline earth metal salt of formic acid.
  • the inducer comprises sodium formate, potassium formate, and/or ammonium formate.
  • an inducer is added by doses or boluses in amount of 0.001-2.0g per 1 L of the culture up to 20 times a day.
  • FDH NAD + dependent formate dehydrogenase enzyme
  • the 50%(w/v) solution of potassium formate is added in amount of 1g/1L of the culture 2 times a day.
  • the method of the present invention allows the production of a heterologous polypeptide or any other added value compound without methanol or with the proviso that no methanol or substantially no methanol, that is, insufficient methanol on its own, is added.
  • the present invention provides a method for producing recombinant proteins and other added value compounds without use of methanol as an inducer. Instead, the method uses a sorbitol feeding and/or an alternative induction strategy for induction gene(s) of interest.
  • the first and/or the second carbon sources can be any carbon source except methanol.
  • the first and/or the second sources comprise a compound selected from the group consisting of alanine, lactate, glycerol, glucose, ethanol, citrate, sorbitol, xylose, trehalose, arabinose, fructose, melibiose, maltose, rhamnose, mannose and raffinose.
  • the batch and fed-batch phases carbon source is glycerol and the induction phase carbon source is sorbitol.
  • the induction pattern of the following promoters were assessed by the measuring the transcription level by means RT-qPCR method: FDH promoter (NAD + -dependent formate dehydrogenase) promoter, the Alcohol oxidase 1 (AOX1 ) promoter, the dihydroxyacetone kinase (DAK) promoter, the dihydroxyacetone synthase 2 (DAS2 ) promoter, the Formyl-glutathione dehydrogenase (FGH) promoter, the Fructose 1,6-bisphosphate aldolase (FBA) promoter, the Peroxisomal membrane signal receptor PTS1 (PEX5) promoter, the Alcohol dehydrogenase 2 (ADH2) promoter.
  • Constitutive GAP Glyceraldehydes-3-phosphate dehydrogenase promoter's induction level was used as a reference.
  • the yeast cultures Pichia pastoris GS115 and KM71h strains were grown under repressed, derepressed and induced conditions.
  • the repressed conditions suggest a repressive carbon source in a media, which makes the involved promoters repressed, for example glycerol.
  • the derepressed conditions suggest a non-repressive carbon source in the media, for example sorbitol or any depleted carbon source, which switches the involved promoter to derepressed state.
  • Induced conditions suggest the addition of a compound inti the media, called an inducer, which makes the involved promoters induced.
  • the culture of P. pastoris strain GS 115 was inoculated from YPD plate into the shacking flask with 10ml of liquid YPD. The culture was growth overnight and reinoculated in a following way:
  • RNA was extracted with RNeasy kit (Qiagen, Germany) according to the manufacturer's protocol.
  • Reverse transcription was done with the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, USA).
  • Specific primers for each appropriate gene: FDH, FGH, DAK, DAS2, AOX1 , FBA, PEX5 and ADH2 were used for RT-PCR to assess the level of their transcription and thus the induction level of the respective promoter.
  • the SYBR green method was used for RT-PCR, with the SYBR ® Green qPCR master mix (Bio-Rad, USA) according to the manufacturer's protocol.
  • GAP gene D-glyceraldehyde 3'-phosphate dehydrogenase was used as a standard reference gene.
  • YN medium (+histidine) 6.7g of Yeast Nitrogen Base with Ammonium sulphate; 20mg L-histidine; bring to 1 1 with distilled water.
  • the codon optimized genes of hEGF (SEQ ID NO:14), hSOD3 (SEQ ID NO: 15),hLF (SEQ ID 16) and RBD (SEQ ID NO:21) were cloned to the standard plasmid pL_FDH with the NheI and PacI cloning sites, yielding the plasmids pL hEGF, pL_hSOD3 and pL_hLF respectively, where the GOI (gene of interest) - hEGF or hSOD3 or hLF or RBD were fused with ⁇ MF secretion tag downstream of the inducible FDH promoter but upstream of the GOI.
  • Fig. 2 schematically illustrates the construct containing the GOI and its integration within the yeast genome. Resistance to G418 antibiotic was used for the selection of the transformed clones. Thus, after the transformation the yeast culture was plated onto YPD plates with G418 (0.8mg/ml). The integration of the respective GOI was verified with PCR using forward primer annealing to 3' of pAOX1 and reverse primer annealing to 5' of the GOI.
  • At least 10 clones were grown in the shaking flasks.
  • Selected clones of the recombinant strains GS115/pL_hEGF, GS115/pL_hSOD3, GS115/pL_hLF and GS115/pL_RBD were inoculated into 10 ml of BMGY medium, incubated in temperature controlled orbital shaker for 24 hours at 29°C, 270 RPM.
  • Pre-grown culture was then spun down at 2000g, washed in distilled water divided into two aliquots. Each aliquot was re-inoculated into 10 ml of BMFSY medium, incubated for another 72 hours at 29°C, 270 RPM in the shaking flasks under inducible conditions.
  • the cultures were induced for 3 consecutive days with a daily dose of the final concentration of 0,2%(w/v) potassium formate + 1%(w/v) sorbitol.
  • YPD liquid 10g of Yeast extract; 20g of Peptone; 20g Dextrose; bring to 1 L with distilled water.
  • YPD agar 10g of Yeast extract; 20g of Peptone; 20g Dextrose; 20g Agar; bring to 1 L with distilled water.
  • BMGY 10g of Yeast extract; 20g of Peptone; 100 ml of 1 mM of Potassium phosphate buffer; 6.7g of Yeast Nitrogen Base with Ammonium sulphate; 10g of Glycerol; bring to 1 L with distilled water.
  • BMSFY 10g of Yeast extract; 20g of Peptone; 100 ml of 1 mM of Potassium phosphate buffer pH 6.0; 6,7g of Yeast Nitrogen Base with Ammonium sulphate; 20g of Sorbitol; 0,5g of Potassium formate; bring to 1 l with distilled water.
  • the recombinant strains of Pichia pastoris GS115/pL_hEGF, GS115/pL_hSOD3, GS115/pL_hLF were used to carry out the fermentation to produce hEGF, hSOD3 and hLF respectively. Fermentation was carried out in a temperature-controlled fermenter (10L working volume) to maintain the temperature at 28°C. The pH of the medium throughout the fermentation was controlled automatically using a pH probe, a controller (New Brunswick, BioFlo 3000) and a computer with Biocommand batch software (Eppendorf AG) was used to monitor and control the fermentation.
  • BSM reduced basal salts medium
  • PTM1 trace salts 50mg/L optionally
  • kanamycin 50mg/L optionally
  • the ingredients (per 1 liter) of BSM are listed in Table 2.
  • the culturing or each strain was carried out at 25°C and a dissolved oxygen (DO) content in the medium at a level of 20% or higher.
  • the pH during fermentation was maintained at 6.5 for secreting protein into the medium and for optimal growth by titrating a solution of ammonium hydroxide into the fermentation vessel.
  • the agitation rate was maintained in the range from about 500 rpm to about 1000 rpm to maintain the above-mentioned oxygen concentration in the medium.
  • Aeration rate was carried out to provide about 0.1 to 1.0 volume of oxygen (in liters) per volume of fermentation culture (in liters) per minute (vvm), so as to maintain the above-mentioned dissolved oxygen concentration (DO) in the medium.
  • a minimum amount of Antifoam A cat.
  • glycerol and sorbitol were used at variable rates as the first carbon source and the second carbon source respectively.
  • glycerol was used as the first carbon source to accumulate cell mass
  • sorbitol was used as the second carbon source to sustain cell growth and for inducing protein expression. Table 1.
  • BSM Reduced Basal Salts Medium
  • PTM1 trace salts Twelve (12) ml of filter-sterilized PTM1 trace salts was added to 1 L of BSM medium. The ingredients of PTM1 trace salts (per liter) are listed in Table 3. Table 2. PTM1 trace salts (per 1 liter) Cupric sulfate-5H2O 6.0 g Sodium iodide 0.08 g Manganese sulfate-H2O 3.0 g Sodium molybdate-2H2O 0.2 g Boric Acid 0.02 g Cobalt chloride 0.5 g Zinc chloride 20.0 g Ferrous sulfate-7H2O 65.0 g Biotin 0.2 g Sulfuric Acid 5.0 ml Water to a final volume of 1 liter
  • OD 600 optical density
  • DO dissolved oxygen
  • the second carbon source feeding rate was adjusted in response to DO levels.
  • the level of the carbon source in the culture is an important determinant for protein induction. For example, changes in the DO concentrations (DO spikes) can be used to determine whether all the glycerol is consumed from the culture before adding the second carbon source e.g., sorbitol. Monitoring the level of carbon source ensures that the sorbitol feed does not over accumulate in the fermenter.
  • DO spikes changes in the DO concentrations
  • sorbitol can be used to determine whether all the glycerol is consumed from the culture before adding the second carbon source e.g., sorbitol.
  • Monitoring the level of carbon source ensures that the sorbitol feed does not over accumulate in the fermenter.
  • Fermentation started with the preparation of a seed culture flask that was used as an inoculum.
  • a flask containing a total of 5 mL of BMGY media was inoculated with 50 uL of glycerol stock of either GS115/pL_hEGF or GS115/pL_hSOD3 or GS115/pL_hLF.
  • the inoculate was grown at 29 °C, by shaking the flask at 250-300 rpm for 16-24 hours, until the optical density of the culture at 600nm (OD 600 ) was 2-6.
  • This initial culture was sub-cultured into a second flask containing 5 mL BMGY media for an additional 24 hrs. On day 3, the second flask was sub-cultured into a 2-liter flask containing 200 mL BMGY media for another 16-24 hours, or until the OD 600 of the culture was 2-6.
  • This 200 mL culture served as the inoculum for the fermenter.
  • a fermenter containing 4L of BSM was sterilized prior to inoculation with the yeast culture. After sterilization, the medium was cooled, and the temperature set to 28°C. DO and pH probes were calibrated according to the manufacturer protocol (Mettler Toledo ⁇ Germany). The medium was agitated with the Rushton impeller at 500RPM and higher and aerated at 1.0 vvm using compressed air to bring the DO of the medium to levels suitable for fermentation. The pH of the medium was adjusted to 6.5 using ammonium hydroxide prior to inoculation, followed by the aseptic addition of 4.35 ml of PTM1 trace salts per liter of fermentation medium.
  • kanamycin was added to the medium at a final concentration of 100 ug/ml.
  • This medium was inoculated using 200 mL of yeast culture of OD 600 at 5.0-6.0.
  • the DO of the culture (medium + yeast cells) in the fermenter was measured following inoculation and was recorded as nearly 100%. After the fermentation started, DO was monitored and controlled by the controlling unit of the fermenter using PID (Proportional-Integrative-Derivative) algorithm. If the DO level of the culture dropped below 20%, agitation was increased to bring the DO level of the culture above 20%.
  • PID Proportional-Integrative-Derivative
  • pH was also monitored and controlled by the controlling unit of the fermenter using PID (Proportional-Integrative-Derivative) algorithm, and adjusted by titrating the culture with the 30% (v/v) solution of ammonium hydroxide by the controlling unit.
  • PID Proportional-Integrative-Derivative
  • the wet cell weight at this stage, after the glycerol fed-batch stage was in the range from about 90 g/liter to about 150 g/liter.
  • glycerol Once the glycerol provided in the fed-batch phase was consumed, cell biomass was further increased by initiating a steady feed of glycerol.
  • the glycerol feed was initiated using a 50% w/v solution of glycerol containing 12 ml PTM1 trace salts per liter of glycerol.
  • the feed rate was set to 18.15 ml/hr /liter of the initial fermentation volume.
  • Glycerol feeding was carried out for about four hours or longer (see below), until the wet cell weight was about 300 g/liter.
  • the level of expressed protein was found to depend in part on the wet cell weight of the cell pellet from the glycerol feeding stage of fermentation. The length of the glycerol feeding phase, therefore, was varied to optimize protein yield.
  • the carbon source was switched from a first carbon source, glycerol, to a second carbon source, sorbitol.
  • a transition from glycerol to sorbitol was carried out using a "mixed feed" of glycerol and sorbitol initially.
  • glycerol feeding was slowly decreased from a rate of 18.15 ml/hr/L of culture medium to 0 ml/hr/L of culture medium over a period of 2 hours and the sorbitol feeding rate is slowly increased from 0 ml/hr/L of culture medium to 2.57 ml/hr/L of culture medium over the same 2 hours.
  • the transition from a glycerol feed to a sorbitol feed was carried out at a rate that did not cause any significant spikes or drifts in the pH of the culture.
  • Other surrogate measures of cell growth and cell health were also monitored during the transition phase.
  • the inducer 50% (w/v) solution of potassium formate was added to the cell culture medium at the amount of 2ml/L of culture medium every 12 hours.
  • the entire sorbitol fed-batch phase lasted approximately 72 hours with a total of approximately 0.75 L sorbitol fed per liter of initial volume.
  • the cell density increased during the sorbitol fed-batch phase to a final level of 350 to 500 g/liter wet cells.
  • the expression construct which was integrated to the production strain genome, contained GOI fused with HA-tag at its C-terminus for detection and visualizing purposes and cloned downstream of the promoter FDH.
  • hEGF, hSOD3, hLF or RBD was expressed and secreted into the media during fermentation, it could then be detected on Western blot with anti-HA-tag antibodies.
  • the strain engineering including molecular cloning, transformation, PCR selection was done according to the standard protocols ( Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001 ).
  • the supernatant was harvested by centrifugation (10,000g for 5 min) of the vials containing an aliquot of the culture. SDS-PAGE was done with the 10ul of supernatant followed by the wet transfer to the PVDF membrane according to standard manufacturer's protocol (BioRad, USA). The membrane was then incubated with first anti HA-antibodies (cat. no sc-7392, Santa Cruz Biotechnology, USA) and then with anti-mouse goat antibodies (cat. No. G-21040, Invitrogen, USA) according to the standard protocol (Invitrogen, USA). The membrane was stained with the Pierce TM ECL Plus Western Blotting Substrate kit (cat. No. 32132, ThermoFisher Scientific, USA). The supernatant from a methanol-induced cultures was used as a reference signal ( FIG. 3-6 ).
  • n/a Promoter FGH (S-hydroxymethyl-glutathione hydrolase) SEQ ID No. 5 n/a Promoter DAK (dihydroxyacetone kinase) SEQ ID No. 6 n/a Promoter FBA2 (fructose 1,6-bisphosphate aldolase) SEQ ID No. 7 n/a Promoter PEX5 (peroxisomal membrane signal receptor PTS1) SEQ ID No. 8 n/a Promoter ADH2 (alcohol dehydrogenase 2) SEQ ID No. 9 n/a Promoter CAT1 ( catalase) SEQ ID No. 10 n/a Promoter DAS1 (Dihydroxyacetone synthase 1) SEQ ID No.

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WO2017109082A1 (fr) * 2015-12-22 2017-06-29 Technische Universität Graz Cellule de levure

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Title
"Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SINGH ANAMIKA ET AL: "The Mut+ strain of Komagataella phaffii (Pichia pastoris) expresses PAOX1 5 and 10 times faster than MutS and Mut- strains: evidence that formaldehyde or/and formate are true inducers of PAOX1.", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 104, no. 18, 6 August 2020 (2020-08-06), pages 7801 - 7814, XP037226411, ISSN: 0175-7598, [retrieved on 20200806], DOI: 10.1007/S00253-020-10793-8 *
TYURIN O. V. ET AL: "Deletion of the FLD gene in methylotrophic yeasts Komagataella phaffii and Komagataella kurtzmanii results in enhanced induction of the AOX1 promoter in response to either methanol or formate", MICROBIOLOGY, vol. 84, no. 3, 1 May 2015 (2015-05-01), US, pages 408 - 411, XP093002705, ISSN: 0026-2617, Retrieved from the Internet <URL:http://link.springer.com/article/10.1134/S0026261715030212/fulltext.html> DOI: 10.1134/S0026261715030212 *
VOGL THOMAS ET AL: "Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris", AMB EXPRESS, vol. 10, no. 1, 25 February 2020 (2020-02-25), XP093003617, Retrieved from the Internet <URL:http://link.springer.com/article/10.1186/s13568-020-00972-1/fulltext.html> DOI: 10.1186/s13568-020-00972-1 *

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